Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.     

The regulation of mitotic spindle function

The process of mitosis includes a series of morphological changes in the cell in which the directional movements of chromosomes are the most prominent. The presence of a microtubular array, known as the spindle or mitotic apparatus, provides at least a scaffold upon which these movements take place. The precise mechanism for chromosome movement remains obscure, but new findings suggest that the kinetochore may play a key role in chromosome movement toward the spindle pole, and that sliding interactions between or among adjacent microtubules may provide the mechanochemical basis for spindle elongation. The physiological regulation of the anaphase motors and of spindle operation either before or after anaphase remains equally elusive. Elicitors that may serve as controlling elements in spindle function include shifts in cytosolic calcium activity and perhaps the activation or inactivation of protein kinases, which in turn produce changes in the state of phosphorylation of specific spindle components.     

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs

Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.     

Primitive mitotic mechanisms.

Unorthodox mitotic mechanisms are reviewed and their contribution to the understanding of evolution of the orthodox mitotic apparatus is considered. Dinoflagellates and hypermastigote flagellates are of particular significance because the microtubular mitotic apparatus is entirely extranuclear with the nuclear membrane persisting through mitosis. Chromosomes are attached to the nuclear membrane. In hypermastigole flagellates early kinetochore separation is on the nuclear membrane without any contribution from microtubules. In dinoflagellates the chromosomes are also attached to the nuclear membrane, but at least in some species cytoplasmic microtubules connect to the attachment site. In Syndinium the attachment site resembles a typical kinetochore, but is inserted in the nuclear membrane. A similar kinetochore is found in certain Radiolaria, but with an intranuclear spindle apparatus the association with the nuclear membrane is no longer necessary and has been lost. Mitosis in the yeast Saccharomyces is essentially orthodox, though chromosomes do not condense. No kinetochores are seen, but a single microtubule makes direct contact with the 20 nm chromatin fiber of each chromosome and shortens during anaphase. About 5-10 microtubules are continuous between the spindle pole bodies and form the elongating central spindle.     

Polo-like kinase controls vertebrate spindle elongation and cytokinesis

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.     

Chromosome movement in mitosis requires microtubule anchorage at spindle poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindle sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G₂ nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.Key words: mitotic chromosomes, Xenopus, egg extracts, intracellular scaling, spindle, embryogenesis, cell division     

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.     

Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts

During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis.     

Effects of heavy water (D2O) on the length of the mitotic period in developing sea urchin eggs

The sequence of mitosis in sea urchin eggs was investigated in the presence and absence of D2O. Direct observations of living cells under a polarizing microscope and observations with fixation-staining procedures were used. The duration of mitosis was extended by the presence of D2O. The slight extension of anaphase was due to elongation of the spindle in D2O, but the period from prophase to metaphase was clearly prolonged in the deuterated condition. These results indicate that D2O does not suppress anaphase chromosome movement, but does affect prometaphase and delays the alignment of chromosomes on the equatorial plane of the mitotic spindle at metaphase. The stability of the isolated mitotic apparatus against Ca ions and low temperature also was investigated. There was no difference in the deterioration of isolated spindle birefringence under normal and deuterated conditions. The implications of these results are discussed in relation to the enhancement effect of D2O on the volume and birefringence of the living mitotic spindle.     

Proteome analysis of the human mitotic spindle

The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. Malfunctioning of the spindle can lead to chromosome missegregation and hence result in aneuploidy, a hallmark of most human cancers. Despite major progress in deciphering the temporal and spatial regulation of the mitotic spindle, its composition and function are not fully understood. A more complete inventory of spindle components would therefore constitute an important advance. Here we describe the purification of human mitotic spindles and their analysis by MS/MS. We identified 151 proteins previously known to associate with the spindle apparatus, centrosomes, and/or kinetochores and 644 other proteins, including 154 uncharacterized components that did not show obvious homologies to known proteins and did not contain motifs indicative of a particular localization. Of these uncharacterized proteins, 17 were tagged and localized in transfected mitotic cells, resulting in the identification of six genuine spindle components (KIAA0008, CdcA8, KIAA1187, FLJ12649, FLJ90806, and C20Orf129). This study illustrates the strength of a proteomic approach for the analysis of isolated human spindles and identifies several novel spindle components for future functional studies.     

Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.     

Aurora kinase inhibitor ZM447439 blocks chromosome-induced spindle assembly, the completion of chromosome condensation, and the establishment of the spindle integrity checkpoint in Xenopus egg extracts

The Aurora family kinases contribute to accurate progression through several mitotic events. ZM447439 ("ZM"), the first Aurora family kinase inhibitor to be developed and characterized, was previously found to interfere with the mitotic spindle integrity checkpoint and chromosome segregation. Here, we have used extracts of Xenopus eggs, which normally proceed through the early embryonic cell cycles in the absence of functional checkpoints, to distinguish between ZM's effects on the basic cell cycle machinery and its effects on checkpoints. ZM clearly had no effect on either the kinetics or amplitude in the oscillations of activity of several key cell cycle regulators. It did, however, have striking effects on chromosome morphology. In the presence of ZM, chromosome condensation began on schedule but then failed to progress properly; instead, the chromosomes underwent premature decondensation during mid-mitosis. ZM strongly interfered with mitotic spindle assembly by inhibiting the formation of microtubules that are nucleated/stabilized by chromatin. By contrast, ZM had little effect on the assembly of microtubules by centrosomes at the spindle poles. Finally, under conditions where the spindle integrity checkpoint was experimentally induced, ZM blocked the establishment, but not the maintenance, of the checkpoint, at a point upstream of the checkpoint protein Mad2. These results show that Aurora kinase activity is required to ensure the maintenance of condensed chromosomes, the generation of chromosome-induced spindle microtubules, and activation of the spindle integrity checkpoint.     

Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol

Fluorescently labeled tubulin was quickly incorporated into the mitotic apparatus when injected into a live sand dollar egg. After a rectangular area (1.6 X 16 microns) of the mitotic spindle was photobleached at metaphase or anaphase by the irradiation of a laser microbeam, redistribution of fluorescence was almost complete within 30 sec. The photobleached area did not change in shape during the redistribution. During the period of redistribution, the bleached area moved slightly toward the near pole at metaphase and anaphase (means: 1.6 and 1.8 micron/min, respectively). These results indicate that redistribution was not due to the exchange of tubulin subunits only at the ends of microtubules but to their rapid exchange at sites along the microtubules in the bleached region. Furthermore, treadmilling of tubulin molecules along with the spindle microtubules possibly occurred at the rate of 1.6 micron/min at metaphase. Birefringence of the mitotic apparatus increased with a large increase in both the number and length of astral rays shortly after taxol was injected. However, the microtubules did not all seem to elongate at the same rate but appeared to become equalized in length. Chromosome movement stopped within 60 sec after the injection. Centrospheres became large and the labeled tubulin already incorporated into the centrospheres was excluded from the enlarged centrospheres. Shortly after the labeled tubulin was injected following the injection of taxol, it accumulated in the peripheral region of the centrospheres, suggesting that microtubules first assembled at this region. Fluorescently labeled tubulin in the mitotic apparatus in the egg after injection of taxol was redistributed much more slowly after photobleaching than in uninjected eggs.     

Meiotic maturation of mouse oocytes in vitro: association of newly synthesized proteins with condensing chromosomes.

The nature, intracellular distribution, and role of proteins synthesized during meiotic maturation of mouse oocytes in vitro have been examined. Proteins synthesized during the initial stages of maturation are concentrated within the nucleus (germinal vesicle) and become intimately associated with the condensing chromosomes. Inhibition of protein synthesis during this period does not prevent germinal vesicle dissolution or chromosome condensation, but meiotic progression is blocked reversibly at the circular bivalent stage. A protein is synthesized during meiotic maturation of the mouse oocyte which exhibits several of the characteristics of the very lysine-rich histone, FI; this and other histones are phosphorylated during the initial stages of maturation. These results are discussed in relation to studies of meiotic maturation of oocytes from non-mammalian species and chromosome condensation in both oocytes and mitotic cells.     

Mitotic apparatus formation and cleavage induction by micromanipulation of the nucleus and centrosome: The centrosome forms a spindle together with only the chromosomes at a short distance

We micromanipulated the nucleus and centrosomes in the zygote of the starfish, Asterina pectinifera, in order to investigate their roles in mitotic apparatus formation and cleavage induction. The zygote cleaved without spindle formation when its nucleus was removed. When one or two centrosomes were transplanted, they formed asters in the recipient cell, which cleaved into three or four blastomeres so that each blastomere might contain one centrosome or aster. When one centrosome was removed, a half-spindle formed in the manipulated cell, which did not cleave until the other centrosome was duplicated. When both centrosomes were removed, no microtubular structures such as the spindle and the aster appeared in the manipulated cell, which failed to cleave. These results indicate that two centrosomes or more in the cell induce cleavage with or without the nucleus and that one centrosome or less does not induce cleavage. It is also concluded that the centrosome(s) together with the nucleus forms a half-spindle or bipolar spindle. However, from the experiments of nucleus transplantation and displacement, spindle formation is found to depend on the distance between chromosomes and centrosomes. The half-spindle formed when the distance from the centrosome to the chromosomes was shorter than 22 microns; on the other hand, when the distance was longer than 22 microns, the nucleus remained apart from the aster, which means that the functional range of the astral microtubule's ability to engage chromosomes was 22 microns from the centrosome.     

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis, but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindles sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes, but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G2 nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.     

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self-reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes.     

Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint

The spindle checkpoint prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein CDC20 until the chromosomes are properly aligned and attached to the mitotic spindle, at which point MCC disassembly releases CDC20 to activate the APC/C. Once the APC/C is activated, it targets cyclin B and securin for degradation, and the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined the effect on the MCC and APC/C activation. Using this approach we found that 2 phosphatase inhibitors, calyculin A and okadaic acid (1 μM), caused MCC dissociation and APC/C activation leading to cyclin A and B degradation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.